In the fermentation of ethanol, the ultimate step in the pathway is the reduction of acetaldehyde to ethanol by the enzyme alcohol dehydrogenase. The product ethanol must then be separated from the reaction mixture, an energy-intensive process which may account for 63% of the total energy use involved in the fermentation process [Kalter et al (1981) ERDA report #81-7 pp. 136-144]. If the reaction pathway is altered, however, to inhibit the reduction of acetaldehyde to ethanol, the acetaldehyde produced might be collected as an attractive alternative to ethanol.
Pure acetaldehyde has a boiling point of 20.8.degree. C., whereas ethanol boils at 78.5.degree. C. Thus, at temperatures typical for mesophilic fermentation, acetaldehyde may be more readily stripped from solution than ethanol. In addition to the advantage of having greater volatility than ethanol, acetaldehyde does not form an azeotrope with water. In January, 1987, 99% acetaldehyde had a market place value nearly 11/2 times that of synthetic 100% ethanol and a value approximately twice that of fermentation ethanol. Acetaldehyde is quite reactive and can be used to produce a variety of compounds such as acetic acid, acetic anhydride, ethyl acetate, butanol, and pyridines. Acetaldehyde also has uses in the food industry as a flavor additive. It is listed as a GRAS (Generally Recognized As Safe) substance by the U.S. Food and Drug Administration and delivers a "fresh" and/or "fruity" flavor to foods such as meats, fruits, breads, spices, vegetables, and dairy products, as well as to candies and chewing gums. Since acetaldehyde is both volatile and reactive, dry acetaldehyde delivery systems are currently being developed for use in dry flavors and instantized foods [Byrne et al (1984) Food Technology 38:57-61].
Previous research on the commercial production of acetaldehyde by biological means has been limited to the reoxidation of biologically produced ethanol back to acetaldehyde. Kierstan (1982)Biotech. Bioeno. 24:2275-2277, conducted a preliminary study on the feasibility of using a free enzyme system to oxidize aqueous solutions of ethanol to acetaldehyde. This enzyme system consisted of the alcohol oxidase from Candida boidinii and a catalase. Similarly, U.S. Pat. No. 4,481,292 was issued for the production of acetaldehyde from ethanol using an enzyme complex containing alcohol dehydrogenase, NADH, flavine mononucleotide, and a catalase. Using this system, a conversion of 10-20% of the ethanol was obtained; after nine hours, acetaldehyde was produced at a level of 2.5 g/l of solution. Armaldehyde strong et al, (1984) Biotech. Lett. 6:183-188 have researched the use of whole cells of Candida utilis for the conversion of ethanol to acetaldehyde. The maximum accumulation of acetaldehyde occurred at a level of 6.5% ethanol in solution, with 3.5 g/l acetaldehyde accumulating in batch culture after 5 hours of growth. No increase in acetaldehyde was noted upon additional incubation. Production of acetaldehyde by this method, however, must be carefully regulated so as to limit the conversion of acetaldehyde to acetic acid. The Electrohol process developed by Meshbesher (U.S. Pat. No. 4,347,09) electrochemically converts fermentation alcohol to acetaldehyde. In an assessment of the process, Trevino (1985) USDOE File #DE85016220, determined that yields of 93% or greater must be obtained in order for it to be competitive with the current ethyene-based technology of acetaldehyde manufacture. The Electrohol process is most efficient with a feed stream of 95% ethanol. As the ethanol concentration in the feed stream is reduced, the efficiency of this process drops considerably.
Zvmomonas. mobilis is known to produce acetaldehyde in the presence of oxygen [Schreder et al (1934) Biochem Z. 273:223-242]. This is due to increased NADH oxidase activity resulting in the decreased availability of NADH for the reduction of acetaldehyde to ethanol by alcohol dehydrogenase. In addition, Z. mobilis apparently does not have an aldehyde dehydrogenase to oxidize acetaldehyde to acetic acid (Bringer et al, 1984, Arch. Microbiol. 139:376-381). Alcohol dehydrogenase mutants of Z. mobilis showing increased levels of acetaldehyde production have previously been isolated using allyl alcohol as a selective agent Wills et al 1981, Arch. Bioch. Biophy. 210:775-780. FIG. 1 depicts the effects of oxygen and allyl alcohol upon glucose metabolism in Z. mobilis.
The general principles of the fermentation are including growth kinetics, the isolation, preservation and improvement of microorganism, media requirements, sterilization, development of inocula fermente design and control, aeration and agitation and the like are well known; see for example "Principles of Fermentation Technology", by Stanbury and Whitaker, Pergamon Press, 1984. Likewise, the growth characteristics, medium requirements and the like of Zvmononas mobilis are well known.